Project Summary/Abstract Acinetobacter baumannii (AB) is a nosocomial, multi-drug resistant pathogenic gram-negative bacterium. It is a serious threat among immunocompromised individuals and for patients in intensive and post-operative care units. AB can infect a wide range of anatomic sites including the respiratory tract, bloodstream, wounds and the urinary tract. Its long-term persistence on abiotic surfaces, such as medical devices, and resistance to disinfectants and antibiotics exacerbate the potential of this bacterium as a dangerous pathogen. Moreover, the high prevalence of infection and associated multi-drug resistance, leaves few, if any, antimicrobial treatment options. Accordingly, the World Health Organization (WHO) lists AB as the number one priority among the bacterial pathogens for which new antimicrobials are urgently needed. However, despite its clinical importance, relatively little is known about the molecular basis of AB persistence in the environment, or its mechanisms of pathogenesis. To address these questions, we have identified, by an in vivo transposon-sequencing screen, the full set of genes required by AB during bloodstream infection. Genes predicted to be involved in the metabolism (biosynthesis and catabolism) of the signaling molecule c-di-GMP (cdG), a master regulator of biofilm formation, were among the genes identified. By using a genetic approach in both Escherichia coli and AB, we identified two functional genes; one involved in the biosynthesis, and one in the catabolism. The overall objective of this application is to determine the contribution of the cdG in both persistence in the environment and pathogenesis of AB. Our central hypothesis is the cdG plays a critical role in the infectious cycle of AB by coordinating its transition between the environment and the host, and vice-versa. To test this central hypothesis, we are proposing the following aims: 1) Assess the role of the cdG in persistence and resistance to environmental stresses and colonization of the bloodstream; and 2) Characterize the regulatory networks controlled by cdG. Taken together, this project will lay the foundations in our much-needed understanding on the mechanisms by which AB persists in the environment and infects its host. Furthermore, this work has the potential to identify novel drug targets to both treat AB infections and prevent its persistence on abiotic surfaces. Finally, this knowledge could also be broadly applicable and be used to treat other pathogens that employ a similar infection cycle to AB.